G-proteins are heterotrimeric GTP-binding proteins that, via their association with cell-surface receptors, relay external signals from biologically active molecules including growth factors, cytokines, neurotransmitters and hormones to a variety of cellular effectors.
G-proteins have been shown to mediate the processes of cell growth, proliferation, signal transduction, membrane trafficking and cytoskeletal interactions. They do so by cycling between an active GTP-bound form and an inactive GDP-bound form. Regulation of the on/off rates of nucleotides for G-proteins is controlled by two classes of molecules; GTPase activating proteins (GAPs) which hydrolyze GTP and guanine nucleotide exchange factors (GEFs) that activate G-proteins by catalyzing the replacement of bound GDP with GTP.
ADP ribosylation factors (ARFs) are a subfamily of small monomeric G-proteins, belonging to the Ras superfamily, which regulate critical vesicular traffic pathways. ARFs initiate the secretion of proteins through the endoplasmic reticulum and Golgi by binding to the donor membrane, which in turn, recruits coatomers, leads to bud formation and eventual vesicle release. ARFs also regulate endocytosis, synaptic vesicle formation, peroxisome biogenesis, and phospholipase D activation (Exton, Eur. J. Biochem., 1997, 243, 10-20; Exton, Physiol. Rev., 1997, 77, 303-320; Roth and Sternweis, Curr. Opin. Cell Biol., 1997, 9, 519-526; Schurmann et al., J. Biol. Chem., 1999, 274, 9744-9751; Vaughan and Moss, Adv. Exp. Med. Biol., 1997, 419, 315-320).
A small family of cytosolic adapter proteins have been isolated and shown to function as guanine nucleotide exchange factors for the ARF proteins. These include cytohesin-1 (Liu and Pohajdak, Biochim. Biophys. Acta., 1992, 1132, 75-78), cytohesin-2 (Chardin et al., Nature, 1996, 384, 481-484), ARNO3 (Franco et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 9926-9931; Klarlund et al., Science, 1997, 275, 1927-1930) and EFA6 (Franco et al., Embo J., 1999, 18, 1480-1491). All of the members of this family contain conserved domain structures that include a pleckstrin homology (PH) domain responsible for signal-dependent phospholipid binding (Hemmings, Science, 1997, 275, 1899) and a Sec7 domain that mediates guanine nucleotide exchange. Disclosed in patent application EP 0763597 are peptides encoding the pleckstrin homology domain of cytohesin-1 and cytohesin-2 as well as DNA encoding the peptides and the full-length proteins, cells that express the peptides, and the use of the peptides to treat various disorders, such as atherosclerosis, dissemination of hematopoietic tumors and in the suppression of the immune system subsequent to organ transplantation (Kolanus and Ostner, 1997). Also disclosed are DNA molecules that hybridize to the cytohesin domains under stringent conditions (Kolanus and Ostner, 1997).
Cytohesin-2 (also known as as PSCD2, ARNO for ARF nucleotide binding site opener, mSec7-2 and ARF exchange factor) was the second ARF GEF member to be isolated and was shown to promote guanine nucleotide exchange on ARF1 (Paris et al., J. Biol. Chem., 1997, 272, 22221-22226), ARF3 (Chardin et al., Nature, 1996, 384, 481-484) and ARF6 (Frank et al., J. Biol. Chem., 1998, 273, 23-27).
While cytohesin-1, the first member of the family to be identified, is expressed predominantly in hematopoietic cells, cytohesin-2 is more ubiquitously expressed and localized to the plasma membrane rather than the Golgi (Frank et al., J. Biol. Chem., 1998, 273, 23-27). Activation of ARF1 by cytohesin-2 requires the presence of phospholipids and a decrease in local magnesium concentration (Paris et al., J. Biol. Chem., 1997, 272, 22221-22226) further supporting the localization of cytohesin-2 to the plasma membrane. Vendateswarlu et al. have shown that the translocation of cytohesin-2 occurs in an insulin-dependent manner in murine 3T3 L1 adipocytes and that this translocation requires the enzyme, phosphatidylinositol 3-kinase (Venkateswarlu et al., Curr. Biol., 1998, 8, 463-466). However, others have shown that cytohesin-2 is predominantly cytosolic in HeLa cells and that overexpression of cytohesin-2 inhibits the secretory pathway of alkaline phosphatase. The effects were accompanied by disassembly of the Golgi and thus demonstrate that cytohesin-2 functions in the Golgi to endoplasmic reticulum transport pathway (Monier et al., J. Cell Sci., 1998, 111, 3427-3436).
Acting as an exchange factor for ARF6, which has been shown to regulate the assembly of the actin cytoskeleton, and in conjunction with protein kinase C, cytohesin-2 functions as a critical link between cell-surface receptors and the actin cytoskeleton (Frank et al., Mol. Biol. Cell, 1998, 9, 3133-3146). Treatment of HeLa cells overexpressing cytohesin-2 with protein kinase C inhibitors resulted in the redistribution of cytohesin-2 and actin filaments into membrane protrusions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of cytohesin-2 and strategies aimed at modulating cytohesin-2 function have involved the use of molecules that block upstream entities such as protein kinase C and mutants lacking either the PH or sec7 domains. However, these strategies are untested as therapeutic protocols. Consequently, there remains a long felt need for agents capable of effectively inhibiting cytohesin-2 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of cytohesin-2 expression. The present invention provides compositions and methods for modulating cytohesin-2 expression.